The Nitrate Transporter Family Protein LjNPF8.6 Controls the N … · The Nitrate Transporter...

The Nitrate Transporter Family Protein LjNPF8.6Controls the N-Fixing Nodule Activity1

Vladimir Totev Valkov,a Alessandra Rogato,a Ludovico Martins Alves ,a,2 Stefano Sol,a,3 Mélanie Noguero,b

Sophie Léran,b,4 Benoit Lacombe,b and Maurizio Chiurazzia,5

aInstitute of Biosciences and Bioresources, Institute of Biosciences and Bioresources (IBBR), ConsiglioNazionale delle Ricerche, 80131 Napoli, ItalybBiochimie et Physiologie Moléculaire des Plantes, Centre National de la Recherche Scientifique Unité Mixte deRecherche/Institut National de la Recherche Agronomique/SupAgro/Université de Montpellier, Montpelliercedex 1, France

ORCID IDs: 0000-0002-3811-0913 (V.T.V.); 0000-0002-0373-9076 (A.R.); 0000-0001-9924-3093 (B.L.); 0000-0003-2023-9572 (M.C.).

N-fixing nodules are new organs formed on legume roots as a result of the beneficial interaction with soil bacteria, rhizobia. Thenodule functioning is still a poorly characterized step of the symbiotic interaction, as only a few of the genes induced in N-fixingnodules have been functionally characterized. We present here the characterization of a member of the Lotus japonicus nitratetransporter1/peptide transporter family, LjNPF8.6. The phenotypic characterization carried out in independent L. japonicusLORE1 insertion lines indicates a positive role of LjNPF8.6 on nodule functioning, as knockout mutants display N-fixationdeficiency (25%) and increased nodular superoxide content. The partially compromised nodule functioning induces two strikingphenotypes: anthocyanin accumulation already displayed 4 weeks after inoculation and shoot biomass deficiency, which isdetected by long-term phenotyping. LjNPF8.6 achieves nitrate uptake in Xenopus laevis oocytes at both 0.5 and 30 mM externalconcentrations, and a possible role as a nitrate transporter in the control of N-fixing nodule activity is discussed

Nitrate (NO32) and ammonium (NH

4

+) represent themain forms of inorganic nitrogen source for plantgrowth and metabolism, with NO3

2 being the largely

dominant supply form in temperate climates (Millerand Cramer, 2005). Higher plants possess two NO3

2

transport systems to cope with a wide range of externalconcentrations, the low-affinity transport system(greater than 0.5 mM) and the high-affinity transportsystem (less than 0.5 mM), both including constitutiveand inducible types of transport (Tsay et al., 2007). Inhigher plants, low-affinity transport system proteinsare represented mainly by the Nitrate Transporter1/Peptide Transporter Family (NPF), which includes alarge number of genes (53 members in Arabidopsis[Arabidopsis thaliana] and 80 in rice [Oryza sativa]),divided in eight subfamilies and able to transportdifferent substrates (Léran et al., 2014). To date, ni-trate transport activity has been reported for 17 outof 53 NPF proteins in Arabidopsis (Corratgé-Faillieand Lacombe, 2017), with AtNPF6.3 being the onlyexception, as it displays dual affinity for nitrate in thehigh and low concentration ranges (Liu et al., 1999).Dual affinity for NO3

2 uptake in Xenopus laevis oocytesalso has been reported for the Medicago truncatulaMtNPF6.8 (Morère-Le Paven et al., 2011) and MtNPF1.7(previously named Numerous Infection and Polyphe-nolics/Lateral root-organ Defective; NIP-LATD) pro-teins (Bagchi et al., 2012). Moreover, Mtnpf1.7 knockout(KO) mutant plants display more defective lateral rootresponses in planta at low KNO3 concentrations than athigher concentrations, indicating a high-affinity trans-port physiological function (Bagchi et al., 2012). Thespecificity of the spatiotemporal patterns of AtNPFexpression and their regulatory profiles ensure nitrate

1 This work was supported by grants from the Italian Ministry ofEducation (Progetti di Rilevanza Nazionale, PRIN 2010/2011, PRO-ROOT, Prot. 20105XLAXM), from Rete delle Biotecnologie in Campa-nia, Progetto Bio Industrial Processes – BIP – CUP B25C13000290007,from the Institut National de la Recherche Agronomique (ContratJeune Scientifique Ph.D. Fellowship to S.L.), and from the Agence Na-tionale de la Recherche (ANR-14-CE34-0007-01-HONIT with a postdoc-toral fellowship to M.N.). L.M.A. was supported by a Short-TermFellowship from the European Molecular Biology Organization.

2 Current address: Buchmann Institute for Molecular Life Sciences,Goethe University Frankfurt, Max-von-Laue-Strasse 15, 60438 Frank-furt, Germany.

3 Current address: CEINGE Biotecnologie Avanzate, Napoli, Italy;and Department of Biology, University of Naples Federico II, 80145Napoli, Italy.

4 Current address: Interactions Plantes Microorganismes Environ-nement, Unité Mixte de Recherche Centre de Coopération Internatio-nale en Recherche Agronomique pour le Développement/Institut deRecherche pour le Développement/Université Montpellier, 911 ave-nue Agropolis, BP64501, 34394 Montpellier cedex 05, France.

5 Address correspondence to [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Maurizio Chiurazzi ([email protected]).

All authors critically revised the article; V.T.V., A.R., L.M.A., M.N.,S.S., and S.L. performed research and analyzed data; B.L. and M.C.designed research; M.C. conceived the project and wrote the article.

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http://orcid.org/0000-0002-3811-0913http://orcid.org/0000-0002-3811-0913http://orcid.org/0000-0002-3811-0913http://orcid.org/0000-0002-3811-0913http://orcid.org/0000-0002-3811-0913http://orcid.org/0000-0002-0373-9076http://orcid.org/0000-0002-0373-9076http://orcid.org/0000-0002-0373-9076http://orcid.org/0000-0002-0373-9076http://orcid.org/0000-0002-0373-9076http://orcid.org/0000-0001-9924-3093http://orcid.org/0000-0001-9924-3093http://orcid.org/0000-0001-9924-3093http://orcid.org/0000-0001-9924-3093http://orcid.org/0000-0001-9924-3093http://orcid.org/0000-0003-2023-9572http://orcid.org/0000-0003-2023-9572http://orcid.org/0000-0003-2023-9572http://orcid.org/0000-0003-2023-9572http://orcid.org/0000-0003-2023-9572http://orcid.org/0000-0002-3811-0913http://orcid.org/0000-0002-0373-9076http://orcid.org/0000-0001-9924-3093http://orcid.org/0000-0003-2023-9572http://crossmark.crossref.org/dialog/?doi=10.1104/pp.17.01187&domain=pdf&date_stamp=2017-10-25mailto:[email protected]://www.plantphysiol.orgmailto:[email protected]://www.plantphysiol.org/cgi/doi/10.1104/pp.17.01187

uptake from soil, long-distance transport within theplant body, and distribution from source to sink tissues(Krapp et al., 2014; Noguero and Lacombe, 2016).

It iswell known that nitrate alsoplays a role as a signalingmolecule involved in the control of many physiologicalprocesses, including gene regulation (Wang et al., 2004)and root development (Walch-Liu et al., 2006). A crucialrole in the nitrate signaling pathway governing root sys-tem architecture and modulation of the expression ofmany genes has been demonstrated for AtNPF6.3, whichfunctions as a nitrate transceptor (Ho et al., 2009; Krouket al., 2010). In particular, the control exerted by AtNPF6.3on lateral root development, in response to different ex-ternal nitrate concentrations, ismediated by its action as anauxin transport facilitator (Krouk et al., 2010; Bouguyonet al., 2015). Plant NPF members encompass proteins ca-pable of transporting different substrates other than ni-trate, such as di/tripeptides, amino acids, glucosinolates,malate, auxin, abscisic acid (ABA), GA3, and jasmonic acid(Frommer et al., 1994; Jeong et al., 2004; Waterworth andBray, 2006; Krouk et al., 2010; Kanno et al., 2012; Nour-Eldin et al., 2012; Saito et al., 2015; Tal et al., 2016). Inparticular, the multitransport feature recently reported forsome NPF proteins displaying phytohormone transportcapacities may suggest additional roles played by theseproteins in regulatory cross talk linking different physio-logical signals (Krouk et al., 2010; Kanno et al., 2012; Chibaet al., 2015; Saito et al., 2015; Tal et al., 2016). However, thedifferent transport capacities are distributed among theeightNPF subclades identified inplants (Léran et al., 2014),as sequence homologies do not correlate with substratespecificity and the determination of the transported sub-strate cannot be determined from the sequence data alone.

Symbiotic nitrogen fixation (SNF) is part of a multi-step mutualistic relationship, mainly restricted to le-gumes, in which plants provide a niche (represented byroot nodule organs) and fixed carbon to the microor-ganism partner (Rhizobium spp.) in exchange for fixednitrogen. The establishment and functioning of an ef-fective SNF consists of the reciprocal recognition ofsymbiotic partners, penetration, stimulation of corticalcell division (nodule primordium), invasion of dividedcells, differentiation of the endosymbiont, N fixation,and nodule senescence. As for the root system, nitrate,both as nutrient and signal, plays a regulative role in thenodulation program, and high external concentrationsinhibit different steps of SNF, although themechanismsinvolved are still controversial (Carroll and Gresshoff,1983; Carroll and Mathews, 1990; Fujikake et al., 2003;Barbulova et al., 2007; Omrane and Chiurazzi, 2009;Jeudy et al., 2010). The addition of 5 mM nitrate quicklystops nodule growth, and this effect seems to be linkedto a decrease in photoassimilate supply to growingnodules (Fujikake et al., 2003). Furthermore, N-fixationactivity is almost completely lost after a short exposureto high nitrate concentrations (Schuller et al., 1988;Vessey and Waterer, 1992), and several hypotheseshave been offered to explain such a strong impact ofnitrate on nodule activity (Vessey and Waterer, 1992;Minchin, 1997; Naudin et al., 2011; Cabeza et al., 2014).

In N-fixing nodules, bacteria that enter root nodulecells are surrounded by a plant-derived membrane,the peribacteroidal membrane (PBM), which enclosesthe intracellular bacteria in a symbiosome. Inside thesymbiosome, bacteria differentiate into bacteroidswith the ability to fix atmospheric N2 via nitrogenaseactivity. A primary nutrient exchange across the PBMis the transport of carbon energy as products of plantphotosynthates to bacteroid in exchange for fixednitrogen. N fixation is an energy-intensive processthat also requires oxygen for respiration to generateATP and reducing equivalents for the reduction of N2to NH3. At the same time, as bacteroid nitrogenase isinactivated by oxygen, a microaerophilic conditionmust be maintained in rhizobia-containing nodulecells. This is achieved by limiting the rate of oxygeninflux through the outer uninfected cell layers of thenodule (Witty andMinchin, 1998) and by maintaininghigh rates of respiration in mitochondria and bacte-roids of invaded cells (Bergensen, 1996). High respi-ration rates are ensured mainly by the presence atmillimolar concentrations of the high-affinity oxygen-binding protein leghemoglobin, which delivers oxy-gen efficiently to mitochondria and bacteroids forrespiration while buffering free oxygen at the re-quired level (Appleby, 1984). This extremely high rateof respiration in the invaded nodule cells is the mainreason for reactive oxygen species (ROS) generation,whose steady-state concentrations must be strictlycontrolled, as these represent not only toxic by-productsof aerobic metabolism but also key signals for nodula-tion. Nodule-specific metabolic pathways are completedby redox reactions involved in the control of concentra-tions of ROS generated inN2-fixing nodules (Evans et al.,1999; Hernandez-Jimenez et al., 2002; Puppo et al., 2005;Becana et al., 2010; Matamoros et al., 2013). The distinctmetabolic pathways of N2-fixing nodules reflect changesin gene expression for related metabolic enzymes.Genome-wide transcriptomic analyses have allowed theclassification of genes induced in nitrogen-fixing nod-ules, and among these, a significant percentage of genesencoding for transporter proteins have been found(Colebatch et al., 2004; Hogslund et al., 2009; Takanashiet al., 2012). NPF proteins are largely represented in thiscategory of transporters, and at least eight membershave been recently subclassified as nodule-induced (NI)genes in Lotus japonicus (Valkov and Chiurazzi, 2014).

Here, we report the functional characterization ofLjNPF8.6, an NI gene that plays a specific positive roleon nodule functioning controlling nitrogenase activityand nodular ROS content.

RESULTS

LjNPF8.6 Expression Is Induced Progressively inN-Fixing Nodules

We previously reported the identification of a largeL. japonicus NPF family consisting of more than 70 mem-bers, 39 of which have a complete sequence that can be

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retrieved from the L. japonicus whole-genome sequenceresource (Sato et al., 2008; Valkov and Chiurazzi 2014;http://www.kazusa.or.jp/lotus/). Transcriptomic dataprovided throughgene-specific andGeneChip approaches(Hogslund et al., 2009; Criscuolo et al., 2012; Takanashiet al., 2012) allowed the identification of a subclass ofeight NPF genes with a clear-cut NI level of expression(Valkov and Chiurazzi, 2014). Among these, the geneLj3g3v2681670.1 (genomic assembly build 3.0, classi-fied as chr2LjT15I01.230.r2.d in build 2.5) has beensubclassified in clade 8 and provisionally namedLjNPF8.6 (Valkov and Chiurazzi, 2014), which encodesfor a 561-amino acid protein with a molecular mass of62.4 kD.In order to further characterize the profile of expres-

sion of LjNPF8.6, wefirst analyzed the distribution of theLjNPF8.6 transcript in different organs of L. japonicusby quantitative reverse transcription (qRT)-PCR. Seed-lings were germinated on Gamborg B5 medium with-out N sources, inoculated with Mesorhizobium loti, andRNA extracted from different organs after 4 weeks. TheLjNPF8.6 gene is expressed strongly in mature nodules,with an amount of transcript about 10-fold higher thanin roots, whereas it is barely detectable in stems, leaves,and flowers (Fig. 1A). LjNPF8.6 expression also has beentested through a time-course experiment in roots ofL. japonicus inoculated with M. loti at 1 week after sow-ing. The LjNPF8.6 expression pattern is not induced inroots at early times after M. loti inoculation when com-pared with the well-known early symbiotic markerNODULE INCEPTION (LjNIN) gene (Schauser et al.,1999). Moreover, a progressive increase of the amount ofLjNPF8.6 transcript is detected in nodule tissue at 10 and28 d after inoculation (Fig. 1B). This profile of induction,starting after the onset of N fixation and induced pro-gressively during nodule maturation, resembles that ofthe late nodulin genes, suggesting an involvement innodule functioning rather than development.

Isolation of LORE1 Insertion Null Mutants andPhenotypic Characterization

To determine the in vivo function of LjNPF8.6, threeindependent LORE1 insertion mutants have been iso-lated from the LORE1 line collection (Fukai et al., 2012;Urba�nski et al., 2012; Malolepszy et al., 2016). Lines53155, 49638, and 19899, bearing retrotransposon in-sertions in the second and third exons (Fig. 2), havebeen genotyped by PCR, and plants homozygous forthe insertion event into the LjNPF8.6 gene were selectedand transferred to the plant chamber for seed produc-tion. End-point reverse transcription-PCR revealed nodetectable LjNPF8.6 mRNA in mature nodules of ho-mozygous plants screened from lines 53155, 49638, and19899; hence, these can be considered null mutants(Supplemental Fig. S1). Initially, two individual ho-mozygous mutant plants from each line were selectedfor analyses, and because their growth phenotypes didnot differ significantly, the data obtainedwith the selected

individual mutants were pooled in this study. The initialphenotyping of the three LORE1 mutant lines includedmeasurements of shoot lengths and fresh weights of4-week-old plants with and without inoculation withM. loti. As shown in Figure 3, in the absence of N sources(noN) or in the presence of KNO3 concentrations (100mM,1 mM) compatible with full nodulation capacity, thethree lines did not present significant differences,when compared with wild-type plants, in terms ofshoot biomass and nodule number (Fig. 3). In orderto test whether LjNPF8.6, which also is expressed at asignificant level in the root tissue, could be involved inthe nitrate-dependent inhibitory pathways controlling thenodule formation process, nodulation capacity also wastested in the presence of high external concentrations ofKNO3 (10 mM). As expected, the number of nodules isreduced strongly in L. japonicus wild-type plants (85%;Barbulova et al., 2007), and an identical inhibitory re-sponse is observed in the Ljnpf8.6 mutants (Fig. 3C).

However, a careful analysis of phenotypes of the in-oculated plants allowed us to detect a visible accumu-lation of anthocyanin, conferring deep purple color, instems of mutant plants in symbiotic conditions whencompared with wild-type plants (Fig. 4, A and B). The

Figure 1. LjNPF8.6 transcriptional regulation. A, Expression in differentorgans. RNAs were extracted from wild-type plants grown on GamborgB5 derivative medium without N source at 4 weeks post inoculation(wpi). Mature flowers were obtained from L. japonicus plants propa-gated in the growth chamber. B, Time course of expression in root andnodule tissues afterM. loti inoculation. RNAs were extracted from rootsof wild-type seedlings grown in N starvation conditions at differenttimes after inoculation (R0, 24 h, and 72 h) and from young (10 d postinoculation) and mature (28 d post inoculation) nodules. Expressionlevels are normalizedwith respect to the internal control ubiquitin (UBI)gene and plotted relative to the expression of flowers (A) and R0 (B).White bars, LjNPF8.6; gray bars, LjNIN. Data bars represent means andSD of data obtainedwith RNA extracted from three different sets of plantsand three real-time PCR experiments.

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anthocyanin accumulation starts to be easily detectablein inoculated Ljnpf8.6 plants at 17 to 20 d after inocu-lation, and the spreading of pigments increases pro-gressively up to the third internode (40%–45% of thestem length) at 4 wpi, whereas in wild-type plants,traces of pigmentation are observed only at the base ofthe stem structure (Fig. 4, A and B). A quantitativeanalysis performed through anthocyanin extractionfrom stem tissues at 4 wpi revealed a content 210% to250% higher in nodulated KO than in wild-type plantsgrown either under noN or 1mMKNO3 conditions (Fig.4C). A significant systemic increase of anthocyanincontent also is revealed in roots of mutant plants inoc-ulated with M. loti (Supplemental Fig. S2A). Con-versely, uninoculated mutant plants did not displayanthocyanin accumulation in stems and roots, as noquantitative differences were detected in wild-type andmutant plants grown in the presence of 1 or 5mMKNO3(Fig. 4C; Supplemental Fig. S2A). Line 53155 wasanalyzed only for plants inoculated on 1 mM KNO3conditions and not utilized further for phenotypiccharacterization, because of the segregation of thenod2phenotypedue to the additional LORE1 insertion inthe CERBERUS gene (Yano et al., 2009). However, theidentical phenotype displayed by the 53155, 49638, and19899 lines confirms that the LORE1 insertion in theLjNPF8.6 gene is the causal mutation of the increasedanthocyanin content observed exclusively in symbi-otic conditions. In addition, heterozygous plants forthe LORE1 insertion in the LjNPF8.6 gene, isolated inthe three lines, did not display high levels of antho-cyanin in the stem (data not shown).

Ljnpf8.6 Nodule Mutants Display Nitrogenase ActivityDeficiency under Permissive Low-Nitrate Conditions,Associated with a Long-Term Shoot BiomassReduction Phenotype

The accumulation of anthocyanin is a clear marker ofplant response to different stress conditions such as lowN availability (Diaz et al., 2006). In order to investigatewhether the anthocyanin accumulation detected in theLjnpf8.6 mutant in symbiotic conditions is correlatedwith reduced nodule functionality, we comparedN-fixation activity in nodules of wild-type and mutantplants at 4 wpi. A significant 25% decrease of acetylenereduction activity (ARA) is detected in nodules ofLjnpf8.6 mutants grown either in the absence of N or inthe presence of 1 mM KNO3 (Fig. 5A). However, the

reduction of N-fixation capacity detected in the Ljnpf8.6KO genetic background is not correlated with any evi-dent shoot phenotype other than anthocyanin accu-mulation during in vitro growth, whichmust be limitedto a short period of analysis (4 wpi; Fig. 3). In order tocheck whether a more severe shoot biomass phenotypecould be displayed by the Ljnpf8.6mutants, 4-week-oldnodulated plants were transferred in growth conditionscompatible with long-term phenotypic analyses. Wefirst tried to transfer the 4-week-old nodulated plantsto pots filled with inert material, but the phenotyp-ing analyses were biased by a random, genotype-independent stress response due to bad adaptationto the new conditions of growth. Conversely, thetransfer of nodulated plants to hydroponic condi-tions minimizes this unpredictable plant phenotype,and all the plants could be scored for reliable shootphenotypes after another 4 weeks. Figure 5, B and C,shows the striking shoot biomass-deficient pheno-type displayed by the Ljnpf8.6mutants 4 weeks aftertransfer to hydroponic conditions (8 wpi). The twomain representative phenotypes observed in all theinoculated mutant plants are stunted shoots withpale green and/or abscised leaves (Fig. 5, B and C).

Ljnpf8.6Mutant Nodules Display Superoxide Overproduction

In M. truncatula, the NIP/LATD protein has been as-sociated, through the characterization of the weak allelicmutant nip-3, with defects in bacteria release or prolif-eration within NI cells, where fewer bacteria are ob-served (Teillet et al., 2008). Therefore, we tested whetherthe deficient N fixation activity observed in the Ljnpf8.6nodules (Fig. 5A) is associated with a reduced invasioncapacity. Seedlings grown in N starvation conditionswere inoculated with an M. loti strain carrying a consti-tutively expressed hemA::lacZ reporter gene fusion forthe staining of young and mature nodules. As shown inFigure 6, A to D, no differences were observed in thedensity of the invading M. loti strain in wild-type andmutant nodules.

Reduction of N-fixation activity in mature nodulesalso has been associated with oxidative damage pro-voked by ROS overproduction, which can be due tonatural aging or to exposure to different stress condi-tions (Becana et al., 2010). We monitored superoxide(O2

2) production at different stages of nodulation usingthe ROS-reactive dye nitroblue tetrazolium(NBT). Thenascent nodule primordia are strongly stained in both

Figure 2. Exon/intron organization of theLjNPF8.6 gene. Insertion sites and relativeorientations of the LORE1 retrotransposon el-ement in the 53155, 19899, and 49638 linesare indicated.


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the wild type and mutants (Fig. 6, E and F), while, asnodules mature and enlarge in size, the staining inten-sity decreases strongly in both genotypes but remainsmore intense in mutant nodules (Fig. 6, G and H). In60% of wild-type big nodules, the staining is not evendetectable in whole-mount samples, whereas in all thecomparable mutant nodules, this is still clearly visible.In whole-mount samples, NBT staining is confined tothe lenticel structure on the nodule surface (Fig. 6, Gand H). Longitudinal nodule sectioning has confirmedthese differences of staining intensity in the paren-chyma region (Fig. 6, I and J). The quantitative analysis

conducted on mature nodules confirms a significantoverall increase of O

2

2 content in the 49638 line com-paredwithwild-type plants (25%; Fig. 6K). O2

2 contentalso was quantified on root and stem tissues of inocu-lated plants, where no significant differences were ob-served (Supplemental Fig. S2B). These results indicate alocal O2

2 increase in the nodule organ.

LjNPF8.6 Is a Nitrate Transporter

Nitrate transport activity has been reported for 17 outof 53 NPF proteins in Arabidopsis (Corratgé-Faillie andLacombe, 2017), which have been characterized as low-affinity transporters, with the exception of AtNPF6.3/NRT1.1, which is a dual-affinity nitrate transporter (Hoet al., 2009; Krouk et al., 2010). In clade 8 of the plantNPFfamily (Léran et al., 2014), the only NPF membercharacterized up to now as a nitrate transporter whenexpressed in X. laevis oocytes is the rice OsNPF8.9protein, which shares 45% amino acid identity withLjNPF8.6 (Lin et al., 2000). In order to assess whetherLjNPF8.6 encodes a nitrate transporter, in vitro-synthetized LjNPF8.6 complementary RNA (cRNA)was injected into X. laevis oocytes for functional assay.Two days after the injections, oocytes were testedfor nitrate 15NO3 uptake activity at two different nitrateconcentrations at pH 5.5: low (0.5 mM) and high (30mM).LjNPF8.6 cRNA-injected X. laevis oocytes were comparedwith the AtNPF6.3-injected oocytes. Both batches ofoocytes display NPF-dependent 15NO3 accumulationin 30 mM as well as 0.5 mM external nitrate (Fig. 7).Within this range of concentrations, a Michaelis-Mentenfit leads to a Km of 7.8 mM, indicating an LjNPF8.6 low-affinity transport capacity (Supplemental Fig. S3),while ahigh-affinity capacity (low NO3

2 concentrations) rangewas not tested.

The uptake activity observed inX. laevis oocytes (Fig.7) prompted us to test for possible roles of LjNPF8.6associatedwith the nitrate transport function importantfor nodule activity. Therefore, we checked whetherLjNPF8.6 could play a role in the inhibitory pathwayresponsible for the abrupt decrease of nodule activitydescribed after exposure to external high nitrate con-centrations (Schuller et al., 1988; Vessey and Waterer,1992). Wild-type and mutant nodulated plants (4 wpi)were transferred for 3 d in the presence of 10 mM KNO3,and nodule activity was analyzed by ARA. The nitro-genase activity is inhibited at the same level in bothwild-type and Ljnpf8.6 plants, ruling out the hypothesisof LjNPF8.6 involvement in the signaling pathwayinhibiting nodule functioning at high external nitrateconcentrations (Fig. 5A).

Jeong et al. (2004) reported the identification of anNPFgene (AgDCAT1) expressed in nodules ofAlnus glutinosathat encodes for a protein capable of malate transport inheterologous systems. Therefore, we also tested the ca-pacity of LjNPF8.6 to transportmalate, the carbon sourcesupplied to bacteroids for metabolism and nitrogen fix-ation (Day and Copeland, 1991). LjNPF8.6 was cloned

Figure 3. Quantitative analysis of shoot biomass and nodulation ca-pacity of L. japonicus wild-type (wt) and LjNPF8.6 null mutant plants,grown in the presence of different KNO3 concentrations, in symbioticand nonsymbiotic conditions. A, Shoot length per plant. B, Fresh shootweight per plant. C, Nodule numbers per plant. The different KNO3concentrations and, when determined, M. loti inoculations are indi-cated. Bars corresponding to wild-type and different LORE1 plants areindicated. Data bars represent means and SE of measures from threeexperiments (12 plants per experiment per condition). Data in A and Bwere scored 25 d after sowing (21 d after transferring the plants fromwater agar). Data in C were scored 28 d after inoculation.



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into the Escherichia coli expression vector pKK223-3under the control of the tac promoter (Brosius andHoly, 1984), and the resulting plasmid was used totransform the dicarboxylate transport mutant CBT315strain (dctA; Lo et al., 1972). Functional complemen-tationwas tested onM9mediumwith 10mMmalate asthe sole carbon source with or without isopropylthio-b-D-thiogalactoside as an inducer of the tac promoter,and the Arabidopsis gene AtALMT6 (At2G17470),encoding amember of the aluminum-activatedmalatetransporter family, was used as a positive control(Meyer et al., 2011). We did not observe any growth ofthe CBT315 strain transformed with the LjNPF8.6-expressing plasmid, while At2G17470 was able to com-plement the malate transport defect (Supplemental Fig.S4). This negative result suggests that LjNPF8.6 does notencode for a malate transporter, although we cannotexclude that the L. japonicus protein is not correctlyexpressed and/or addressed to the plasma mem-brane in E. coli.

Ljnpf8.6 Mutants Have Unaltered Nitrate Content inDifferent Plant Organs

In order to check whether LjNPF8.6 plays any role inthe uptake of external nitrate and/or the distribution ofthis nutrient to different plant tissues, we compared thenitrate content of different organs in wild-type andLjnpf8.6mutant plants. The analyses were conducted at4 wpi in plants grown in the presence of 1 mM KNO3.The comparison of nitrate content either in roots orleaves of wild-type and mutant plants did not showsignificant differences, confirming that LjNPF8.6 playsa role strictly related to nodule functionality (Fig. 8A).In the microaerophilic condition associated with thenodule environment, the efficiency of N fixation is as-sociated largely with nitrate-dependent respirationpathways in the NI cells (Kato et al., 2003; Meilhocet al., 2010; Horchani et al., 2011). Therefore, we testedwhether the reduced nitrogenase activity observed inthe Ljnpf8.6 mutants in low nitrate-permissive condi-tions (Fig. 5A) might be associated with a different ni-trate content in nodules ofwild-type andmutant plants.The nitrate content was quantified in detached wild-type and mutant nodules of plants inoculated on noN and 1 mM KNO3 conditions and displaying the an-thocyanin accumulation phenotype. The results shownin Figure 8B indicate that, independent of the presenceof external nitrate supply, a significant content of thisnutrient is detectable in nodule tissues and that nosignificant differences are observed on nitrate accu-mulation in nodules of wild-type and mutant plants.However, since the nitrate-dependent respirationpathway for the maintenance of the nodule energystatus becomes more important in hypoxic than innormoxic conditions (Horchani et al., 2011; Hichri et al.,2015), we also tested whether the stressful phenotypedisplayed by the Ljnpf8.6 null mutants at 4 wpi couldqualitatively worsen in hypoxic conditions. Two-week-old wild-type and 46938 plants with the same numberof nodules that do not show any anthocyanin accu-mulation symptoms were transferred in hydroponiccultures to reproduce the water-logged conditions thatprovoke hypoxic stress. Interestingly, mutant plantsgrown for an additional 2 weeks in hydroponic condi-tions show a clear-cut increase of the anthocyanin ac-cumulation phenotype, which is clearly visible throughoutthe length of the stem tissue almost up to the shoot apex(80%–85%of the stem length versus 40%–45%displayed innormoxic conditions; Fig. 4, A and B), whereas wild-typeplants did not show such evidently stressful symptoms(Supplemental Fig. S5).

DISCUSSION

We report here the functional characterization of amember of the L. japonicusNPF family, LjNPF8.6, whichplays a positive role in the symbiotic interactionthrough a nodule-associated function. NPF proteinsrepresent a significant number of the NI transporters,and the qRT-PCR analysis shown in Figure 1 indicates

Figure 4. Analysis of anthocyanin content. A, Three representativewild-type and 49638 plants are shown on the right and left sides of thepetri dish, respectively. B, Higher magnification showing intense purplecolors in the stems of mutant plants (on the left). C, Anthocyanin contentin stems of wild-type (wt) and LORE1 lines. The different KNO3 con-centrations and, when determined, M. loti inoculations are indicated.Bars corresponding to wild-type and different LORE1 plants are indi-cated. Data bars represent means and SE of measures from three ex-periments (12 plants per experiment per condition). Data in C werescored 28 d after inoculation. Asterisks indicate significant differences(P , 0.001) from wild-type levels.


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that LjNPF8.6 is strongly induced in nodule tissue, al-though it cannot be considered a strict late nodulingene, as it is also expressed in root tissues. This result isconsistent with the analysis reported by Hogslund et al.(2009), indicating that most genes functioning in ma-ture nodule also are expressed elsewhere in the plant.The induced profile of LjNPF8.6 expression in nodulesis consistent with the specific symbiotic phenotypesdisplayed by independent KO Ljnpf8.6 LORE1 insertionmutants. In particular, a striking anthocyanin accu-mulation is observed in stems and roots of Ljnfp8.6mutants compared with the wild type only after M. lotiinoculation, as no increase of pigmentation is observedin uninoculated plants grown in the presence of dif-ferent nitrate concentrations (Fig. 4; Supplemental Fig.S2A). This result clearly indicates that the role playedby LjNPF8.6 is strictly associated with the symbiotic

program and is not related to the N nutritional status ofplants linked to external nitrate availability. This is alsoconfirmed by the analyses of nitrate content in rootsand shoots that show no significant differences betweenwild-type and mutant plants (Fig. 8A). The productionof anthocyanin is considered a hallmark of the plantresponse to unfavorable growth conditions (Chalker-Scott, 1999), and N limitation has been reported totrigger different anthocyanin biosynthetic pathwaysand accumulation in various plant tissues (Diaz et al.,2006; Rubin et al., 2009; Kovinich et al., 2014).

In the case of SNF, anthocyanin accumulation in thestem is a symptomnormally exhibited bymutant plantsshowing an impaired N-fixation activity (Krusell et al.,2005; Ott et al., 2005; Bourcy et al., 2013). The anthocy-anin accumulation phenotype is displayed in the inoc-ulated Ljnpf8.6 mutants by 17 to 20 d post inoculation,

Figure 5. A, ARA per nodule weight of wild-type and 49638 plants. The different KNO3 conditions are indicated. Data barsindicate means and SE of three independent experiments (n = 8 plants per experiment). Asterisks indicate significant differences(P, 0.005) between wild-type and 49638 nodules in no-N and 1 mM KNO3 conditions. The asterisk over the bracket across the10 mM condition indicates a significant reduction of ARA activity in nodules of both plant genotypes shifted onto high-nitrateconditions comparedwith nodules of plants maintained on low permissive conditions (P, 0.001). B and C, Representative shootphenotypes of 8-wpi wild-type and 49638 plants transferred onto hydroponic conditions in the presence of 0.5 mM KNO3 at4 wpi. Wild-type and mutant plants were maintained in the same vessels (four vessels, 16 plants).



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after the observed induction of LjNPF8.6 expression innodule tissue (Fig. 1B) and the onset of N fixation, sug-gesting a role in the control of nodule functioning rather

than development. Interestingly, further informationabout the spatial profile of LjNPF8.6 in mature L. japonicusnodules came froma tissue-specific profiling carried out by

Figure 6. Phenotypic symbiotic characterization of Ljnpf8.6mutants. A to D, Histochemical detection of b-galactosidase activityto test M. loti (carrying the hemA::lacZ-expressing plasmid) density in young and mature nodules of wild-type (A and C) and49638 (B and D) plants. E to H, Whole-mount NBT staining for O2

2 anion detection of wild-type (E and G) and 49638 (F and H)nodule primordia and mature nodules. I and J, Sections (100 mm) of wild-type (I) and 49638 (J) mature nodules stained with NBT.Arrows indicate staining in the parenchyma (p) and nodular vascular bundles (vb). K, Quantification of NBT staining in wild-type(wt) and 49638 mutant lines. Data bars represent means and SE of nodules from three independent samples (eight plants perexperiment). Asterisks indicate significant differences from wild-type values (P , 0.05). FW, Fresh weight.


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laser microdissection and microarray analysis, whichrevealed a specific expression of six NI NPF members,including LjNPF8.6, in the central infection zonewhere N fixation takes place (Takanashi et al., 2012). Inparticular, the LjNPF8.6 expression was not detectedin the inner cortex and vascular bundle zones, sug-gesting for this NPF member a function associatedspecifically with N fixation (Takanashi et al., 2012).Consistently, Ljnpf8.6 mutants do not show any dif-ference, comparedwith wild-type plants, in the noduleformation capacity at different KNO3 concentrations orin the early steps of nodule primordia invasion (Figs. 3Cand 6, A–D). The direct involvement of LjNPF8.6 in thecontrol of nodule functionality is demonstrated by theanalysis reported in Figure 5A, where a significantlyreduced quota of N-fixation activity (25%) is measuredin mutant nodules. We also demonstrate that LjNPF8.6does not play any role in the quick transport of externalhigh nitrate concentration to the nodule tissues and/orsensing, which must be involved in the nitrate-dependent N-fixation inhibitory pathway (Fig. 5A;Arrese-Igor et al., 1998; Cabeza et al., 2014).Nitrogen fixation is an extremely expensive process

for legume plants, as root nodules are optional C sinkorgans that exploit large amounts of photosynthateresources. The partially compromised N-fixation ac-tivity displayed by the Ljnpf8.6mutants in the presenceof low-concentration N sources (Fig. 5A) is certainly astressful condition, which is responsible of the antho-cyanin accumulation (Fig. 4; Supplemental Figs. S2Aand S4), but it is still sufficient at 4 wpi to sustain anormal shoot biomass phenotype (Fig. 3, A and B).However, when plants are maintained for a longer timein symbiotic conditions, clear-cut N deficiency symp-toms such as stunted shoots with pale green and/orabscised leaves are clearly displayed by the Ljnpf8.6mutants (Fig. 5, B and C). These phenotypes have beenclassified as Fix+/Fix2, associated with mutant plants

with a less efficient N-fixation activity, which displayN-deficiency phenotypes not as severe as in the fix2mutants (Pislariu et al., 2012).

The reduction and loss of N-fixation activity is asso-ciated with oxidative stress during the natural senes-cence of nodules, and similar correlated phenotypes canbe observed in early senescence induced by exposure tostress conditions (Puppo et al., 2005). Therefore, theN-fixation deficiency and increased O2

2 content phe-notypes displayed in mature nodules of Ljnpf8.6 mu-tants (Figs. 5A and 6K) are strictly associated with eachother, although a cause-effect relationship can be diffi-cult to establish. The pattern of O

2

2 production duringnodule growth and maturation, shown in the time-course experiment displayed in Figure 6, is consistentwith previous reports where NBT staining revealedO2

2 accumulation in nodule primordia of indetermi-nate as well as determinate nodules (Fig. 6, E and F;Santos et al., 2001; Montiel et al., 2016). To our knowl-edge, the pattern of O2

2 accumulation reported inFigure 6, G to J, has never been described in maturedeterminate nodules. The NBT staining indicates apredominant localization on the lenticel structures,characterized as the choke points controlling the gase-ous exchanges in determinate nodules (Frazer, 1942;Pankhurst and Sprent, 1975; Jacobsen et al., 1998; Fig. 6,G and H) and the parenchyma regions (Fig. 6, I and J).Biochemical and transcriptomic analyses indicate that,inmaturedeterminate nodules,most of theROS-generatingprocesses, which occur during natural or early senes-cence induced by exposure to stress conditions, origi-nate in the central infected region and then spreadoutward (Evans et al., 1999; Puppo et al., 2005;Matamoros et al., 2013). Therefore, it is reasonable topredict the O2

2 diffusion and accumulation in lenticelstructures and parenchyma tissue, which are located afew cells apart from the infected zone. Furthermore,we demonstrate that the increased content of O2

2

detected in the mutant plants is restricted to nodulartissue (Supplemental Fig. S2B) and, therefore, that thesystemic pattern of anthocyanin accumulation (Fig. 4;Supplemental Fig. S2A) is likely not associated with adirect action of scavenging (Yamasaki et al., 1996).

Members of the NPF familymay encompass differentputative strategic roles associated with the control ofnodule functioning, as they can transport nitrate, aminoacids, peptides, dicarboxylic acids, and ABA (Frommeret al., 1994; Jeong et al., 2004; Waterworth and Bray,2006; Kanno et al., 2012). Malate is the carbon sourcesupplied to bacteroids for metabolism and nitrogen fixa-tion (Day and Copeland, 1991), and in isolated soybean(Glycine max) symbiosomes, a carrier for monovalentdicarboxylate ions with a higher affinity for malate thanfor succinate has been identified (Udvardi andDay, 1997).Jeong et al. (2004) also reported the identification of anNPF gene (AgDCAT1) expressed in nodules of A.glutinosa encoding for aprotein capable ofmalate transportin heterologous systems, but no further functional char-acterization of AgDCAT1 has been provided. The failureof LjNPF8.6 to complement the malate transport defect of

Figure 7. Functional expression of LjNPF8.6 in X. laevis oocytes in low(0.5 mM) and high (30 mM) external nitrate concentrations. Nitrate up-take is shown in control oocytes (black bars), injected with cRNAs,expressing LjNPF8.6 (white bars) or AtNPF6.3 (gray bars; n = 5–8).Values are means 6 SE.



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the E. coli dctA mutant (Supplemental Fig. S4) does notallow us to conclude that it is not involved in the C sourcesupply to bacteroids (Day and Copeland, 1991), as wecannot exclude a nonfunctional expression of the L. japo-nicus transporter in E. coli. Another function that must betaken into account to explain the critical role played byLjNPF8.6 in nodule activity is related to the peptide/amino acid transport capacity. The transport of thesesubstrates may play a positive role in supplying branched-chain amino acids necessary for bacteroid developmentand persistence (Prell et al., 2009) and/or for support-ing polyamine biosynthesis for nitric oxide production(Gupta et al., 2011). ABA signaling also has been shownto be involved in the control of nodule functionality.However, ABA has been reported to negatively regu-late N fixation in L. japonicus, as its reduced contentobtained in low-sensitivity mutants as well as wild-type plants treated with abamine (a specific inhibitorof 9-cis-epoxycarotenoid dioxygenase) resulted in in-creased N-fixation activity, making unlikely the in-volvement of LjNPF8.6 in the nodular ABA transport/signaling pathway (Tominaga et al., 2009).

The preliminary biochemical characterization ofLjNPF8.6 carried out in our work indicates that it iscapable of achieving nitrate uptake in X. laevis oocytesat high (30 mM) and low (0.5 mM) external nitrateconcentrations (Fig. 7). Importantly, the positive roleplayed by LjNPF8.6 in nodule activity, indicated by thenitrogenase deficiency phenotype displayed by Ljnpf8.6mutants (Fig. 5A), is observed in plants grown in thechronic absence of N or in low permissive KNO3 con-ditions (1 mM or less), which is consistent with thereported capacity of enhancing nitrate uptake in X.laevis oocytes at 0.5 mM external nitrate concentration(Fig. 7). NPF members are represented significantlyin the protein fraction associated with the PBM andcharacterized either in L. japonicus or soybean nod-ules (Wienkoop and Saalbach., 2003; Clarke et al.,2015). In particular, the recent comprehensive samplingreported for PBM proteins in soybean has allowedthe identification of five NPFmembers associated withPBM. Interestingly, the retrieved GmNPF8.6 protein(Glyma02g38970.1) also is a member of clade 8 andshares 69% amino acid identity with LjNPF8.6 (Clarkeet al., 2015). A nitrate flux through the PBM, critical fornodule activity, was proposed previously. Aniontransporters, members of the Major Facilitator Super-family, with a selectivity preference for nitrate, havebeen identified in the PBM of soybean and L. japonicusnodules (GmN70 and LjN70), and their role in thecontrol of ion and symbiosome membrane potentialhomeostasis has been postulated (Udvardi et al., 1991;Vincill et al., 2005). In addition, nitrate transportthrough the PBMhas been proposed to be crucial for thenitrate-nitric oxide respiration process reported innodules in microaerobic conditions, which is importantfor the maintenance of the energy status required fornitrogen fixation in normoxic and hypoxic conditions(Igamberdiev and Hill, 2009; Kato et al., 2010; Sánchezet al., 2010; Horchani et al., 2011). Nitrate in the cytosoland symbiosomes of invaded plant cells is produced atelevated rates (Herold and Puppo, 2005; Meilhoc et al.,2010; Horchani et al., 2011), and our analysis of nitratecontent in nodule tissues confirms the presence of anactive nitrate biosynthetic pathway within nodules in-dependently of external supply (Fig. 8B), which mayjustify the symbiotic phenotypes detected in the Ljnpf8.6mutants even in the absence of external N sources (Figs.4 and 5A). Therefore, LjNPF8.6 might play an active rolein the control of nodule activity by participating innitrate flux through the PBM (Udvardi et al., 1991;Horchani et al., 2011). In this context, the unchangednitrate content observed in whole detached nodulesof wild-type andmutant inoculated plants (Fig. 8B) isexpected, as is the distribution of nitrate betweendifferent compartments of N-fixing nodule cells to beaffected in the Ljnpf8.6 background. Interestingly, wehave observed an increased stressful phenotype as-sociated with anthocyanin accumulation in Ljnpf8.6mutants grown under hydroponic hypoxic conditions,where achieving nitrate reduction into nitrite consti-tutes the main route for nitric oxide biosynthesis

Figure 8. A, Nitrate content of shoots and roots from wild-type and49638 plants grown on 1 mM KNO3 and inoculated with M. loti. Databars represent means and SE from three independent samples (10 plantsper sample). B, Nitrate content in wild-type and 49638 4-week-oldnodules. Data bars represent means and SE of nodules from three in-dependent samples (10 plants per sample). Bars corresponding to wild-type and 49638 plants are indicated. FW, Fresh weight.


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(Supplemental Fig. S5; Horchani et al., 2011; Hichriet al., 2015).

CONCLUSION

To our knowledge, LjNPF8.6 represents the first NPFprotein playing a specific role in nodule functioning, asdemonstrated by the description of different N-deficiency-associated phenotypes displayed by independent KOmutants in symbiotic conditions. Further experiments willbe necessary to interpret its mechanism of action, takinginto account different substrate specificities and the possi-ble functional redundancy of these transporters in thenodule organ.

MATERIALS AND METHODS

Plant Material and Growth Conditions

All experiments were carried out with Lotus japonicus ecotype B-129 F14GIFU (Handberg and Stougaard, 1992; Jiang and Gresshoff, 1997). Plants werecultivated in a growth chamber with a light intensity of 200 mmol m22 s–1 at23°C with a 16-h/8-h day/night cycle. Solid growth medium has the samecomposition as B5 medium (Gamborg, 1970), except that (NH4)2SO4 and KNO3are omitted and/or substituted by different concentrations of KNO3. KCl isadded to the medium to replace the potassium source. The medium containingvitamins (Duchefa catalog no. G0415) is buffered with 2.5 mM MES (Duchefacatalog no. M1503.0250) and pH adjusted to 5.7 with KOH.

For hydroponic cultures, wild-type and mutant nodulated plants aretransferred in the same vessels with derivative B5 liquid medium with 0.5 mMKNO3 or without N sources (six plants per vessel). The medium is renewedevery 4 d, when also the pH is checked and maintained within close limits (5.6–5.8) during the entire growth period.

Mesorhizobium loti inoculation is performed as described by Barbulova et al.(2005). For phenotypic comparisons, unsynchronized seedlings are discardedafter germination. The strain R7A is used for the inoculation experiments and isgrown in liquid TYR medium supplemented with rifampicin (20 mg L21). TheM. loti hemA::lacZ strain was kindly provided by Dr. Jens Stougaard (Universityof Aarhus) and was grown in the same medium supplemented with rifampicinand tetracycline (20 mg L21).

LORE1 Line Isolation

LORE1 lines 30053155, 30049638, and 30019899 (hereafter abbreviated as53155, 49638, and 19899, respectively) were obtained from the LORE1 collection(Fukai et al., 2012; Urba�nski et al., 2012; Malolepszy et al., 2016). The plants inthe segregating populations were genotyped, and the expression of homozy-gous plants was tested with the primers listed in Supplemental Table S1.

Determination of ARA

Detachedrootswith comparable numbersofnodules areplaced inglass vials.The vials are filled with an acetylene-air mixture (C2H2:air = 1:9, v/v). After30 min of incubation at 25°C, the amount of ethylene in the gas phase is de-termined using a gas chromatograph (PerkinElmer Clarus 580).

For the analysis of ARA activity after a shift to high-KNO3 conditions, 4-wpinodulated plants are transferred on slanted petri dishes where roots are placedin a sandwich between two filter papers wet with Gamborg B5 liquid mediumcontaining no KNO3 or 10 mM KNO3. Plants are maintained for 3 d in theseconditions with filter papers wet with 20 mL of liquid medium. After 3 d, ARAactivity is tested as described above.

Estimation of Anthocyanin

Stem tissues from three plants per assay areweighed and then extractedwith99:1 methanol:HCl (v/v) at 4°C. The OD530 and OD657 for each sample are

measured, and relative anthocyanin levels are determined with the equationOD530 – (0.25 3 OD657) 3 extraction volume (mL) 3 1/weight of tissue sample(g) = relative units of anthocyanin per g fresh weight of tissue.

Determination of Nitrate Content

Colorimetric determination of nitrate content in nodule extracts followed theprocedure described by Pajuelo et al. (2002). A total of 200 mL of 5% (w/v)salicylic acid in concentrated sulfuric acid is added to aliquots of 50 mL from thecrude extracts and left to react for 20 min at room temperature. NaOH (4.75 mLof 2 N) is added to the reaction mixtures, and the absorbance is read at 405 nmafter cooling. A calibration curve of known amounts of nitrate dissolved in thestandard extraction buffer is used for analytical determinations. Controls are setup without salicylic acid.

LacZ Activity and Histochemical Localization

L. japonicus roots isolated after M. loti hemA::lacZ infection are first gentlyrinsed in 50 mM KH2PO4 buffer, pH 7.2, and then fixed for 1 h with 1% para-formaldehyde (w/v) and 0.3 M mannitol in 50 mM KH2PO4 buffer, pH 7.2. Thetissues are washed again with 5 mM KH2PO4 buffer, pH 7.2, and the histo-chemical analysis is performed according to Omrane et al. (2009). The stainednodulated roots are photographed with a Nikon microscope using bright-fieldand epipolarization optics.

O22 Staining

For O22 staining, NBT (Sigma-Aldrich) was used. Nodulated roots are

submerged in 1 mM NBT in 0.1 mM potassium phosphate buffer (pH 7.5), vac-uum infiltrated for 30 s, and incubated 30 min at room temperature. NBTstaining solution is removed, and stained roots are washed twice in 80% eth-anol. NBT staining is repeated three times for a total of 10 nodules per experi-ment. For longitudinal sections, 12 nodules from independent experiments aresectioned on 100-mm-thick slices. Samples are photographed with a Nikonmicroscope using bright-field and epipolarization optics. For O

2

2 quantifica-tion, NBT-stained nodules are first ground into a fine powder and dissolved in2 M KOH:dimethyl sulfoxide (1:1.16, v/v) followed by centrifugation at 12,000gfor 10 m. Absorbance at 630 nm is immediately measured and then comparedwith a standard curve plotted from known amounts of NBT in the KOH:di-methyl sulfoxide mix (Ramel et al., 2009). Experiments are conducted on threebiological replicates for a total of 40 nodules per genotype.

Real-Time qRT-PCR

Real-time PCR was performed with the DNA Engine Opticon 2 System (MJResearch) using SYBR to monitor double-stranded DNA synthesis. The pro-cedure is described by Rogato et al. (2008). The UBI gene (AW719589) was usedas an internal standard. The concentration of primers was optimized for everyPCR, and amplifications were carried out in triplicate. The PCR program usedwas as follows: 95°C for 3 min and 39 cycles of 94°C for 15 s, 60°C for 15 s, and72°C for 15 s. Data were analyzed using Opticon Monitor Analysis Softwareversion 2.01 (MJ Research). The qRT-PCR data were analyzed using the com-parative Ct method. The relative level of expression is calculated with the fol-lowing formula: relative expression ratio of the gene of interest is 22DCT, whereDCT = CTGENE 2CTUBI. The efficiency of the LjNPF8.6 primers is assumed to be2. Analysis of the melting curve of the PCR product at the end of the PCR runrevealed a single narrow peak for each amplification product, and fragmentsamplified from total cDNAwere gel purified and sequenced to ensure accuracyand specificity. The oligonucleotides used for the qRT-PCR are listed inSupplemental Table S1.

Plasmid Preparation

The plasmid for the expression inXenopus laevis oocytes was prepared in thefollowing way. cDNA prepared from nodule RNA was amplified with a for-ward primer containing the BamHI site in combination with a reverse primercontaining the EcoRI site (Supplemental Table S1). The 1,713-bp fragment wasdouble digested with BamHI-EcoRI and subcloned into the pGEMHE plasmidcontaining the 59 and 39 untranslated regions of the X. laevis b-GLOBIN gene(Liman et al., 1992), predigested with BamHI-EcoRI to obtain pGEMHE8.6. Thecorrect coding sequence of LjNPF8.6 was verified by sequencing.



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For expression in Escherichia coli, pGEMHE-8.6 was double digested withBamHI-EcoRI and subcloned in pKK223-3 plasmid (Brosius and Holy, 1984) toobtain pKK-8.6. Primers for the subcloning of the Arabidopsis (Arabidopsisthaliana) At2G17470 gene used as a positive control in the complementation testare indicated in Supplemental Table S1.

Functional Analysis of LjNPF8.6 in X. laevis Oocytes

pGEMHE-NPF8.6 was linearized with NheI and capped mRNA transcribedin vitro using the mMessage mMachine T7-ultra Kit (Life Technologies). Oocytepreparation has been described (Lacombe and Thibaud, 1998). Defolliculatedoocytes are injected with 20 ng of cRNA and stored in a modified ND96medium(2mMKCl, 96mMNaCl, 1mMMgCl2, 1.8mMCaCl2, 5mMHEPES, 2.5mM sodiumpyruvate, pH 7.5, supplemented with gentamycin sulfate [50 mg mL21]). Twodays after injection, batches of 10 injected oocytes are incubated in 1 mL ofmodified ND96 solution at pH 5.5 supplementedwith 30 or 5 mM 15NO3 suppliedas K15NO3 for 2 h at 18°C. Oocytes are then rinsed five times in 15 mL of coldmodified ND96 solution. Batches of two oocytes are then analyzed for total Ncontent and atomic percent 15N abundance by continuous-flow mass spectrom-etry, using a Euro-EA Eurovector elementar analyzer coupled with an IsoPrimemass spectrometer (GV Instruments). Oocytes injectedwith AtNPF6.3 cRNA andwater were used as positive and negative controls, respectively. The results arepresented asNPF-dependent nitrate accumulation (total 15N in injected oocytes – 15Nin water-injected oocytes) normalized by the accumulation in 30 mM nitrate.

Complementation of E. coli

E. coli K-12 (DCT) and its dicarboxylate transport mutant strain CBR315(CGSC5269) were obtained from the E. coli Genetic Stock Center (Yale Uni-versity). The phenotypes of the transformed strains were compared on M9medium with malate or Glc as the sole carbon source.

Statistical Analysis

Statistical analyseswere performedusing theVassarStats ANOVAprogram.

Supplemental Data

The following supplemental materials are available.

Supplemental Figure S1. Homozygous plants for LORE1 insertions intothe LjNPF8.6 gene are null mutants.

Supplemental Figure S2. Analyses of anthocyanin content in roots of wild-type and 49638 plants and quantification of NBT staining in roots andstems of wild-type and 49638 inoculated plants.

Supplemental Figure S3. Effects of external nitrate concentration on 15Naccumulation in LjNPF8.6-expressing oocytes.

Supplemental Figure S4. Complementation test of the E. coli dctA mutant.

Supplemental Figure S5. Anthocyanin accumulation phenotype in hydro-ponic conditions.

Supplemental Table S1. Oligonucleotide sequences.

ACKNOWLEDGMENTS

We thank the facility of Integrated Microscopy of the Institute of Geneticsand Biophysics for supporting the microscopy analysis and Stefano Rubino,Sara Salvia, and Danilo Maiello for technical assistance.

Received August 23, 2017; accepted September 15, 2017; published September20, 2017.

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